Aristolochic acid (AA) is a naturally occurring compound found only in Aristolochia plants. Although Aristolochia species have long been used for medicinal purposes, their carcinogenic and nephrotoxic properties were recognized only recently. Significant amounts of AA are present in herbal supplements marketed through the Internet. And, in China and other Asian countries, where herbal remedies are widely used, 100 million people are estimated to be at risk of developing aristolochic acid nephropathy (AAN), a devastating and uniformly fatal disease. Importantly, AA?s unique mutational signature has clearly demonstrated its worldwide association with upper urothelial tract cancers, bladder cancer, renal cell carcinoma, hepatocellular carcinoma and intrahepatic cholangiocarcinoma. In this research, we use a multipronged approach to solve a complex global health problem. Thus, we propose to identify mechanisms of AA-induced carcinogenicity and nephrotoxicity, important causes of morbidity and mortality worldwide. Our guiding hypothesis is that genes involved in bioactivation and inactivation of AA, and its transport to target tissues, act both independently and together to determine individual susceptibility to AA?s toxic effects. Using an integrated human liver-kidney-on-a-chip, we have recently demonstrated that AA activation occurs primarily in the liver. Activated, chemically labile AA metabolites bind tightly to serum albumin and subsequently are transported to the kidney, where they form covalent adducts with proteins and DNA. In Aim 1, we establish pathways of AA metabolism, monitor the pharmacokinetics of AA, and quantify the distribution of AA metabolites, aristolactam (AL)-DNA adducts, and AL-protein adducts among the blood, urine, and target tissues of sensitive and resistant strains of mice. In Aim 2, we extend our human ?organs-on-a-chip? studies and apply mass spectrometric, fluorometric, and plasmon resonance techniques to illuminate the critical role of serum albumin in protecting and transporting activated species of AA. However, although the genotoxic properties of AA account for its carcinogenic effects, the molecular mechanisms of its nephrotoxicity are unknown. We hypothesize that selective binding of AA to proteins in the renal proximal tubule initiates the cytotoxicity underlying AA?s nephrotoxic effects. Thus, in Aim 3, we use novel proteomic techniques, affinity probes, and monoclonal antibodies to explore nephrotoxic events, with the goal of identifying the specific renal tubular proteins involved. Successful completion of this research program will advance significantly our knowledge of mechanisms involved in the dual toxicities of AA. Also, data obtained in this study may provide a basis for establishing individual susceptibility to AA, and will facilitate early diagnosis, prevention, and treatment of AA-induced cancers and chronic renal disease. Given the worldwide exposure to AA, this research has significant implications for global public health. Additionally, the techniques perfected in the three aims of this project will serve as templates for investigating other causes of nephrotoxicity and carcinogenicity.